Opioids are the most effective treatment for pain. Unfortunately, their potency decreases with repeated administration because of the development of tolerance. The broad long-term objective of this research is to improve the treatment of pain by learning how to block the development of tolerance to opioids. Although morphine tolerance has been studied extensively, the neural mechanisms underlying tolerance remain poorly understood. One problem is that opioids act at peripheral, spinal, and supraspinal sites and tolerance may be mediated by different mechanisms at each site. Another problem is that there are several different types of tolerance (behavioral, associative, and non-associative) and different mechanisms may underlie each type. The proposed studies overcome these problems by focusing on tolerance mediated by a brain structure known as the ventrolateral periaqueductal gray (vPAG). The vPAG is of particular interest because it is part of the primary descending pain modulatory system in the brain and the neural circuitry through which vPAG opioids produce antinociception has been well characterized. Moreover, previous research has shown that tolerance develops to administration of morphine directly into the vPAG. Thus, the vPAG provides a unique opportunity to identify both the specific classes of neurons and the mechanisms underlying different types of tolerance to opioids. The first aim of the proposed studies is to determine whether behavioral, associative, and non-associative factors contribute to vPAG mediated tolerance. This will be accomplished by microinjecting morphine into the vPAG in environmental situations that facilitate each type of tolerance. The second aim is to determine whether tolerance is caused by a change in opioid-sensitive GABAergic neurons or output neurons. Opioids in the PAG produce antinociception by inhibiting GABAergic neurons and disinhibiting output neurons. If the mechanism for tolerance resides within opioid-sensitive GABAergic neurons, then tolerance should not develop to microinjection of drugs that act directly on vPAG output neurons. This hypothesis will be tested by examining tolerance to repeated microinjection into the vPAG of the neuroexcitant kainate, the GABA antagonist bicuculline, and the cannabinoid CP-55,940. The proposed studies will provide a foundation for future research examining specific neural mechanisms for different types of vPAG mediated tolerance. In the long term, such knowledge should improve the treatment of pain by allowing therapies that disrupt tolerance to be developed.